The central objective of this study is the elucidation of molecular processes which underlie the functional properties of proteins which form channels in biological membranes. Two channel forming colicins, E1 and B, will be incorporated into planar lipid bilayers to investigate two such problems of general interest: the mechanism of ion transport and selectivity of membrane channels, and the nature of the conformational changes which accompany insertion of proteins into membranes and the gating of channels. Colicin proteins are encoded on extrachromosomal plasmids in host bacterial cells and are produced in large quantities in a water-soluble form. Thus, the primary structures of these proteins are accessible for chemical and genetic manipulation. The channels formed by colicin E1 and colicin B have many functional features by which they may be distinguished. These features argue for their selection for this study. Colicin channels conduct both positively and negatively charged ions. While even the smallest ions are conducted at fairly slow rates, no large ion has yet been identified which behaves as if it were excluded from the channel. We propose to use a series of ionic substances as probes of the channel lumen. In addition to studies of the permeation and block of colicin channels by these ions, the competition between blocking and permeant ions will be examined. These studies will provide information about the channel's steric and electrostatic topography and the types of ionic interactions which are allowed within the channel. Colicin channels can exist in a membrane either as open channels or closed channels. We propose to determine whether any closed channels have structures which span the entire membrane. To do this, we will test the ability of chemical and biological agents to attack the colicin protein from the opposite side of a membrane. The ion selectivities of the colicins depend upon pH. Recent results have suggested that when colicin E1 is initially exposed to a neutrally charged planar bilayer at pH 5, the membrane insertion process traps the protein in a state whose selectivity is characteristic of pH 8. To test this hypothesis, colicin channels will be subjected to cyclical changes in pH while their selectivity is monitored. The demonstration of a time independent hysteresis loop in the selectivity will be sufficient grounds to rule out insertion as the critical event in this phenomenon and have significant implications for the nature of the conformational changes controlling selectivity.